The goal of this research program is to develop new and useful physical mapping procedures that may be applied directly to the human genome. We propose to clone and construct a complete high resolution restriction map of chromosomes IV of Drosophila melanogaster. Unlike most of the drosophila genome that is composed predominantly of unique sequences, the fourth chromosome is littered with repeated elements interspersed among unique stretches of DNA throughout its length. This short period interspersion structure is characteristic of the human genome as well. In this sense, Drosophila will serve as a unique and convenient paradigm for establishing methodologies that may be readily transferred to cloning and sequencing the human genome. Establishing an overlapping set of clones for genomes possessing repetitive sequences requires special caution. It is not sufficient to find merely cross hybridization between the termini of different clones. Sequence relatedness and the presence of pseudogenes are apt to result in erroneous linkages if only this criterion is used. Rather, what is required is the demonstration of substantial homologous overlap as can be most readily obtained by intensive restriction mapping. We have already developed lambda phage, plasmid and cosmid vectors and techniques that allow one to easily and unambiguously construct extremely high resolution restriction maps of cloned DNA fragments. These procedures, when coupled to our rapid oligodeoxynucleotide synthesis protocols as well as to our proposed methods for more efficient cosmid screening and the localization of repeated sequences in cloned DNA, should make megabase walking and restriction mapping an efficient process. Specifically, we intend to accomplish the following. We will construct restriction maps of cosmid clones, starting from the ci region of chromosome IV of Drosophila, using a DNA sequencer adapted to this purpose. Repeated sequences present in the clone will also be located on the map. Unique sequence DNA probes corresponding to the termini of the initiating clone will be synthesized directly on a solid support. These immobilized unique probes will then be used to hybridize complementary cosmid DNA. This process should enrich significantly for cosmid clones contiguous with either end of the initiating clone. Cosmid DNA isolated in this way will then be used to transform E. coli by electroporation. Clones overlapping with the initiating cosmid will be quickly identified. This process will be repeated with the new set of contiguous clones. Methods for walking with YACs are also proposed and these vectors may be integrated into this scheme.